Antibodies with simultaneous subsite specificities to protein and lipid epitopes

ABSTRACT

Antibodies and method of making antibodies, either monoclonal or polyclonal wherein said antibodies have dual or multi-specific binding capacity to more than one type of antigenic epitope. The antibodies have simultaneous or independent recognition subsites to each of the epitopes. Antigenic epitopes include lipids, peptides, proteins, amino acid sequences, sugars and carbohydrates. Monoclonal antibodies and a method of making monoclonal antibodies of the invention include monoclonal antibodies that are broadly neutralizing to HIV-1 or other envelop viruses wherein the monoclonal antibody has subsites that simultaneously recognize protein and lipid epitopes from the virus.

This application is a continuation of and claims priority of U.S. Ser.No. 11/525,574 filed Sep. 22, 2006 which claims priority from U.S.Provisional Application No. 60/772,084 filed Sep. 23, 2005, bothapplications incorporated herein by reference.

This application contains a sequence listing that is the same as thesequence listing in the parent application U.S. Ser. No. 11/525,574,incorporated herein by reference.

GOVERNMENT INTEREST

The invention described herein may be manufactured, used and licensed byor for the U.S. Government.

BACKGROUND OF THE INVENTION

1 Field of the Invention

The present invention relates to a method of making dual specificantibodies. More specifically, the present invention relates to a methodof making antibodies that are dual specific to both (1) amino acidsequences and (2) solid phase lipid structures. The present inventionhas relevance to such important subject matter as making broadlyneutralizing monoclonal antibodies to HIV-1.

2 Brief Description of Related Art

One of the major barriers that have emerged in the development of aneffective HIV-1 vaccine is the difficulty in obtaining neutralizingantibodies that block infection by primary isolates derived from a widecross-section of clades (subtypes). In order to obtain broadlyneutralizing or protective antibodies to HIV-1 it is necessary forantibodies to utilize antigenic epitopes (i.e., molecular recognitionsites for binding of antibodies) that are conserved in the virus or thatare present in the host or target cell in the regions in which the viruseither buds or where binding or fusion with the virus occurs (McMichael& Hanke 2003; Burton et al. 2004). Most mammalian cells have arelatively conserved repertoire of lipids in the lipid bilayer of theplasma membrane, including glyceryl phospholipids, sphingosylphospholipids (mainly sphingomyelin), lysophospholipids,glycosphingolipids, and cholesterol.

The human immunodeficiency virus type 1 (HIV-1) is an enveloped viruswith a lipid bilayer that contains several glycoproteins that areanchored in, or closely associated with, the membrane surface. Theenvelope proteins have complex interactions with the lipids both on thehost cells and on the target cells. The processes of budding from hostcells and entry into target cells occur at sites on the plasma membrane,known as lipid rafts that represent specialized regions that are rich incholesterol and sphingolipids. Although the envelope glycoproteins areantigenic molecules that potentially might be used for development ofbroadly neutralizing antibodies in a vaccine to HIV-1, the developmentof such antibodies that have broad specificities against primaryisolates of virus have been largely thwarted to date by the ability ofthe envelope proteins to evade the immune system through variousmechanisms.

It has been known for more than 20 years that monoclonal antibodies canhave subsite specificities that simultaneously recognize differentepitopes, such as simultaneous recognition of different types ofcarbohydrates; or combinations of carbohydrate and sulfated molecules,or carbohydrates and phosphorylated molecules. These subsites fordifferent epitopes exist simultaneously in the same overall antigenbinding site of the antibody. In our research, we have found polyclonalor monoclonal antibodies to membrane associated lipid antigens that alsocontain subsites that recognize unrelated phosphate or sulfatedmolecules as an epitope. We have also found that numerous membraneassociated protein antigens have subsites that also recognize phosphateand even cross-react with phospholipids. However, this research has notproduced a monoclonal antibody that is broadly neutralizing to HIV-1.

Therefore, an object of the present invention was to make antibodiesthat have dual specific action by recognizing, as antigens or epitopes,both (1) amino acid sequences such as proteins, peptides andpolypeptides and also (2) solid phase lipid structures such as lipids,liposomes and the like so that the antibody will have greater affinityfor these antigens or epitopes at the surface of target organisms orcells. The amino acid sequences and solid phase lipid structures may befrom entities such as viruses, bacteria, cancer cells, hormones or anyother substance that produces an immune response, wherein both (1) and(2) are capable of being recognized individually or together (i.e.,simultaneously) by the antibody.

Another object of the invention was to apply this strategy to obtainantibodies that are broadly neutralizing to HIV-1 because they havesubsites that recognize both protein and lipid or carbohydrate antigenicepitopes that are present either on the virus or on the budding site,receptor site, or fusion site of the plasma membrane.

In the case of HIV-1, this is necessary for the antibody to have dualspecificity with the HIV-1 protein and with the plasma membrane of thehost cell in the vicinity of the HIV-1 virus. In the case of otherentities that produce an immune response, the antibodies will either beto the lipids themselves or to the combined lipid and amino acidsequences. The antibodies will either interfere with the entity throughsteric hindrance, or through conformational changes in the lipids thatwill interfere with the viability of the entity, or that will activatecomplement or other types of innate immunity as an effecter mechanism.

FIG. 4 is a schematic model of the HIV-1 putative trimeric envelopespike. The viral particle 2 is shown inserted into the plasma membrane5. Most of the surface of gp 41 is believed to be occluded by gp120.However, the amino acid sequences of gp41 close to the membrane thathave been identified as binding sites of MABs 2F5, Z13, and 4E10 havebeen suggested to be exposed to antibody binding (Zwick et al., 2001).IgG is shown as 20.

The invention solves the problems associated with the past lack ofability to find antibodies that are broadly neutralizing. In the case ofHIV-1, the invention solves the problem by showing that patterns ofplasma membrane lipids, known as lipid rafts, serve as binding sites notonly for viral interactions with host and target cells, but also aslipids that might be incorporated into HIV-1 to comprise the lipidbilayer of the virus envelope and exploiting this knowledge to producemonoclonal or polyclonal antibodies that recognize these lipids as wellas HIV-1 peptides. This invention will have particular relevance for HIVvaccine research and development, and for the treatment of HIV-1 and forresearch, vaccine development, and treatment of other enveloped viruses.

SUMMARY OF THE INVENTION

The present invention relates to a method of making dual specificantibodies. More specifically, the present invention relates to a methodof making antibodies that are dual specific for binding to both (1)amino acid sequences and (2) organized lipid structures, such as lipidspresent in a lipid bilayer membrane.

The present invention is also directed to a method of making monoclonalantibodies by obtaining liposomes having lipid epitopes similar to thosepresent on HIV-1 and modifying the liposomes by including an adjuvant inthe liposomes, or by injecting the liposomes together with an adjuvant,and such liposomes also contain protein or peptide epitopes from HIV-1virus. The liposomes contain lipid combinations comprising cholesterol,sphingomyelin, charged phospholipids, phosphatidylethanolamine,galactosyl ceramide, or sulfogalactosyl ceramide to name a few of thelipids from the lipid raft region of the plasma membrane. Then theliposomes are inserted into a mammal to produce monoclonal antibodiesagainst the liposomes. The monoclonal antibodies have simultaneousrecognition subsites to lipid epitopes in the liposome and to theprotein of HIV-1 virus.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

The accompanying drawings show illustrative embodiments of the inventionfrom which these and other of the objectives, novel features andadvantages will be readily apparent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the plasma membrane glycosphingolipidmicrodomains as preferential sites of formation of the HIV-1 fusioncomplex;

FIG. 2 is a diagram of the interactions of HIV-1 envelope proteins withplasma membrane lipids during target cell binding (B) and fusion steps(C);

FIG. 3 is a schematic diagram of HIV-1 gp 41 envelope protein (SEQ IDNO: 1);

FIG. 4 is a model of the HIV-1 molecule showing gp 41 at the vicinity ofthe lipid bilayer.

FIGS. 5 a, b, c, d, and e are graphs showing the binding of fivedifferent clones and their recognition capabilities as shown by ELISAand made by the method of this invention;

FIGS. 6 a and 6 b are graphs showing the binding of anti-PIP and 4E10antibodies, respectively, to CL as determined by ELISA and the effectson bindings by soluble haptens;

FIGS. 7 a and 7 b are graphs showing the binding of anti-PIP and 4E10antibodies, respectively, to PIP antigens as determined by ELISA and theeffects on bindings by soluble haptens;

FIG. 8 a is a graph showing anti PIP antibody neutralizing activity; and

FIG. 8 b is a graph showing 4E10 antibody neutralizing activity.

DETAILED DESCRIPTION

The method of the present invention applies to the making of monoclonalantibodies and antibodies that have dual specificity and are broadlyneutralizing. The dual specificity is obtained by making monoclonalantibodies and polyclonal antibodies that recognize both (1) amino acidsequences such as amino acid sequences of one or more of proteins,peptides and polypeptides and (2) organized lipid domains, includingsolid phase lipid structures such as one or more types of lipids,liposomes or the like.

The method involves obtaining an organized lipid structure that haslipid properties that resemble or mimic the lipids found in a particularentity. Entities include viruses, bacteria, hormones, fungi, protozoa,cancer cells, or anything else that produces an antibody when introducedto a mammal. Then added to the organized lipid structure are amino acidsequences that resemble or mimic amino acid sequences from peptideepitopes, polypeptides, proteins or the like. This modified lipidstructure is then inserted into a mammal to induce an immune responsewhich is the production of antibodies that are dual specific to thelipids and amino acid sequences in the modified organized lipidstructure. Optionally and preferred is to also incorporate an adjuvantinto the modified solid phase lipid structure.

Materials and Methods: HIV-1

Murine antibodies were made by injecting mice with liposomes containinglipid A and protein from HIV-1 (either gp 160, gp 120, gp140, or gp 41).The preferred antibody is a monoclonal antibody.

Preparation of Liposomes:

The liposomes were prepared by making a liposome containing one or morelipids found in the lipid bilayer of the plasma membrane of the hostcell in the region of the lipid raft or one or more types of lipidsnormally found in HIV-1. Then the adjuvant, lipid A and the protein fromHIV-1 were inserted into the liposome. The liposomes contain lipidcombinations comprising one or more of cholesterol, sphingomyelin,charged phospholipids, phosphatidylethanolamine, galactosyl ceramide, orsulfogalactosyl ceramide to name a few of the lipids from the lipid raftregion of the plasma membrane. The lipid A and the protein are eitherattached to the surface of the liposomes, or intercalated into theliposomal membrane bilayer, or encapsulated in the aqueous spaces insidethe liposome

The liposomes are easily prepared using methods known in the art and asfound in U.S. Pat. Nos. 5,888,519, 6,093,406, incorporated herein intheir entirety by reference.

The following general methods for manufacturing liposomes have beenpublished, and are incorporated in their entirety by reference:

Swartz, Jr., G. M., Gentry, M. K., Amende, L. M., Blanchette-Mackie, E.J. and Alving, C. R. Antibodies to cholesterol. Proceedings of theNational Academy of Sciences, U.S.A. 85 1902-1906 (1988).

and,

Alving, C. R., Shichijo, S., Mattsby-Baltzer, I., Richards, R. L. andWassef, N. M. Preparation and use of liposomes in immunological studies.Liposome Technology, vol. 3, (Second Edition), (Gregoriadis, G., ed.),CRC Press, Inc., Boca Raton, Fla., pp. 317-343 (1993). Some specificdetails are given below. This describes the preferred liposomecomposition.

Lipids from Avanti (dimyristol-phosphatidylcholine (DMPC),dimyristol-phosphatidylglycerol (DMPG), and cholesterol) dissolved indistilled chloroform were added to 50 ml pear-shaped flasks in 9:1:25(DMPC:DMPG:cholesterol) molar ratio along with Lipid A (Avanti) in afinal concentration of 200 μg/ml. Lipids were deposited as a thin filmunder 0.1 kPa vacuum at 40° C. on a rotaevaporator with 230 rpm. Thelipids were then dried overnight in desiccator. The high cholesterol(71%) liposomes were formed in distilled water then lyophilized for 24hours. Gp140 protein oh HIVIIIB (The Biotech Source) in PBS was added tothe lipids to yield 50 mM phospholipid suspensions, formingmultilamellar liposomes with 100 μg/ml incorporated HIV glycoprotein.All suspensions were stored at 40° C. until injection.

Liposomes within the present invention can be prepared in accordancewith known laboratory techniques. In one preferred embodiment, liposomescan be made by mixing together the lipids to be used, including lipid A,in a desired proportion in a container, e.g, a glass pear-shaped flask,having a volume ten times greater than the volume of the anticipatedsuspension of liposomes. Using a rotary evaporator, the solvent isremoved at approximately 40° C. under negative pressure. The vacuumobtained from a filter pump aspirator attached to a water faucet may beused. The solvent normally is removed within about 2 to 5 minutes. Thecomposition can be dried further in a desiccator under vacuum. The driedlipids are generally discarded after about 1 week because of itstendency to deteriorate with time.

The dried lipids can be hydrated at approximately 30 mM phospholipid insterile, pyrogen-free water by shaking until all the lipid film is offthe glass. The aqueous liposomes can be then separated into aliquots,each placed in a vaccine. vial, lyophilized and sealed under vacuum.

In the alternative, liposomes can be prepared in accordance with otherknown laboratory procedures, e.g., the method of Bangham et al., J. Mol.Biol. 13: 238-52 (1965), the contents of which, are incorporated hereinby reference; the method of Gregoriadis, as described in “Liposomes” inDRUG CARRIERS IN BIOLOGY AND MEDICINE, pp. 287-341 (G. Gregoriadis ed.1979), the contents of which are incorporated herein by reference; themethod of Deamer and Uster as described in “Liposome Preparation:Methods and Mechanisms” in LIPOSOMES (M. Ostro ed. 1983), the contentsof which are incorporated by reference; and the reverse-phaseevaporation method as described by Szoka, Jr. and Papahadjopoulos, in“Procedure for Preparation of Liposomes with Large Internal Aqueousspace and High Capture by Reverse-Phase Evaporation,” Proc. Natl. Acad.Sci. USA 75: 4194-98 (1978). The aforementioned methods differ in theirrespective abilities to entrap aqueous material and their respectiveaqueous space-to-lipid ratios.

Synthetic lipid A can be purchased from available commercial sources,e.g., Calbiochem-Behring (La Jolla, Calif.), List BiologicalLaboratories, Inc. (Campbell, Calif.) and Corixa (formerly, RibiImmunochem Research, Inc.) (Hamilton, Mont.). LPS is similarly availablefrom commercial sources, e.g., Difco (Detroit, Mich.), List BiologicalLaboratories, Inc. and Corixa. When the lipid A is inserted into theliposome, the toxic portion of lipid A which usually causes fever isembedded in the liposome. Therefore, lipid A does not cause a toxicresponse and has been found not to be dangerous. The inventor found thatwhen lipid A is embedded in the liposome and injected in mice, rabbits,or humans, it sends out a signal to the immune system. The immune systemis then immobilized to send out an immune response to anything that isnear the lipid A. In the preferred embodiment of the invention, theimmune system sends out a response that attacks HIV-1 by producingantibodies that simultaneously recognize lipid epitopes and HIV-1protein or peptide epitopes. Lipid A is used as an adjuvant, however,other adjuvants that are known to induce an immune response may also beincorporated into the liposome.

The liposomes contain a variety of lipid compositions that mimic theknown lipid composition of HIV-1 and that also include lipid A as aliposomal adjuvant. An example of such a liposome composition cancontain one or more of phosphatidyl choline (PC),phosphatidylethanolamine (PE), sphingomyelin (SM), phosphatidylserine(PS), Phosphatidylinositol-4-phosphate (PIP) and cholesterol in theapproximate concentrations found in HIV-1 isolated as described in Aloiaet al., Lipid composition and fluidity of the human immunodeficiencyvirus envelope and host cell plasma membrane, Proc. Natl. Acad. Sci. USA1993; 90:5181-5185. Cholesterol can also be increased in the liposomesto optimize the induction of antibodies to cholesterol. The liposomescan also contain galactosyl ceramide (GalCer), sulfogalactosyl ceramide(SGalCer), ceramide trihexoside (CTH), ganglioside GM1 (GM1), organglioside GM3 (GM3), and phosphatidyl glycerol. These syntheticliposomes have lipids that mimic the lipid raft of the plasma membraneand which are also incorporated into the structure of HIV-1

Included within the scope of the present invention is changing orincorporating of one or more of the different lipids mentioned above inthe liposome in order to achieve a desired result. Any of a combinationof the lipids mentioned, or similar types of lipids, or synthetics, arewithin the scope of the invention.

Incorporation of HIV-1 Protein into Liposomes

As indicated above, in addition to the above lipids, the liposomes alsocontain relevant HIV-1 protein or peptide epitopes that bind to orinteract with lipids, including gp160, gp140, or gp41 from HIV-1envelope. The liposomes may also contain nef, either alone or with envantigens, because nef, is known to react with the lipid bilayerparticularly in association with cholesterol. It is generally found thatthe amino acid sequences that are present above the transmembranesequences of gp41 are highly conserved, and the lipids themselves arehighly conserved.

HIV-1 is a spherical, enveloped RNA retrovirus, a lentivirus that fuseswith the plasma membrane of a host cell to insert its genomic RNA. Theenvelope of HIV-1 contains a lipid bilayer that is associated with twoloosely bound glycoproteins, gp120 and gp41. These proteins are createdduring intracellular virus assembly when a precursor protein, gp160, iscleaved to form gp120 and gp41 (FIG. 1A). The gp41 is a trimerictransmembrane protein that is anchored in the lipid bilayer, and duringviral maturation and budding the intraviral end of gp41 is bound tosites located on an N-terminal myristoylated matrix protein (p17) (Yu etal. 1992; Freed & Martin 1995; Hill et al. 1996). As with otherenveloped viruses, the lipid composition of the virus is largelyreflective of the composition of lipid rafts present in the plasmamembrane of the host cell (Aloia et al. 1988 and 1993).

Cells that become infected with HIV-1 undergo cytopathological effectsleading to apoptosis. This may initially occur because arginine residueson the C terminal end of gp41 (fusion peptide) electrostatically bind tophosphatidylserine (PS) or PG or other charged phospholipids on theinner lamella of the plasma membrane of the target cell. Theinteractions of the C terminus of gp41 with PS or PG results in theformation of a lipidic pore, or causes other lipid membrane changes,leading to cellular permeability, cytopathology, apopotosis withmovement of PS from the inner lamella of the lipid bilayer to the outerlamella, and death (Chernomordik et al., 1994; Trommeshauser and Galla,1998; Trommeshauser et al., 2000). After the subsequent acquisition ofviral phospholipids from the host cell plasma membrane by the buddingvirus, the resultant viral lipid bilayer resembles that of apoptoticcells in that PS is present on the outer lamella of the budded viralbilayer (Callahan et al., 2003).

Protein is incorporated into the liposomes when it is included in theaqueous solution that is used to disperse the dried lipids. Theliposomes form automatically and automatically enclose a volume of theaqueous protein solution that was used to disperse the liposomes.

Monoclonal Antibody Production

Monoclonal antibodies were made to the liposomes of the invention byusing techniques well known in the art for making monoclonal antibodiesand as found in U.S. Pat. Nos. 4,885,256 and 6,900,025, incorporated byreference. Monoclonal antibodies were produced that have subsitespecificities both for lipid and amino acid epitopes and that will bindsimultaneously to the lipids and the lipid-associated protein.

Methods for producing and obtaining an antibody are well known by thoseskilled in the art. An exemplary method includes immunizing any animalcapable of mounting a usable immune response to the antigen, such as amouse, rat, goat sheep, rabbit or other suitable mammal. In the case ofa monoclonal antibody, antibody producing cells of the immunized animalmay be fused with “immortal” or “immortalized” human or animal cells toobtain a hybridoma which produces the antibody. If desired, the genesencoding one or more of the immunoglobulin chains may be cloned so thatthe antibody may be produced in different host cells, and if desired,the genes may be mutated so as to alter the sequence and hence theimmunological characteristics of the antibody produced. Fragments ofbinding agents, may be obtained by conventional techniques, such as byproteolytic digestion of the binding agent using pepsin, papain, or thelike; or by recombinant DNA techniques in which DNA encoding the desiredfragment is cloned and expressed in a variety of hosts. Irradiating anyof the foregoing entities, e.g., by ultraviolet light will enhance theimmune response to a multi-epitopic antigen under similar conditions.Various binding agents, antibodies, antigens, and methods for preparing,isolating, and using the binding agents are described in U.S. Pat. No.4,471,057 (Koprowski), U.S. Pat. No. 5,075,218 (Jette, et al.), U.S.Pat. No. 5,506,343 (Fufe), and U.S. Pat. No. 5,683,674(Taylor-Papadimitriou, et al), all incorporated herein by reference.Furthermore, many of these antibodies are commercially available fromCentocor, Abbott Laboratories, Commissariat a L'Energie Atomique,Hoffman-LaRoche, Inc., Sorin Biomedica, and FujiRebio.

Immunization of Mice

The immunization procedure was performed by Biocon Inc. followed thecompany's approved protocol. The animal handling, quarantine measures,monitoring and vaccination of mice were used with maximal safety andminimal pain.

Liposomes were mixed with Freud's adjuvant for immunization. The groupsize to was 5 animals per immunization. They were ear tagged and prebledafter they were released from quarantine. The animals were immunized byintraperitoneal route (IP). Two weeks after immunization one animal wasselected on the basis of the ELISA data screening for the antibodiesagainst to lipids and gp140 protein. The animal was anesthetized, andterminally bled by cardiac puncture. The spleen was removed andprocessed for fusion with myeloma cells. The remaining 4 mice wereboosted at week 3 by the IP with the same liposomal antigen formulation.They were bled at week 5, and the sera were assayed for antibodies tothe antigens. The best responsive mouse was selected and immunized bythe IV route. Four days later, the animal was anesthetized andterminally bled by cardiac puncture. The spleen was removed andprocessed for making hybridomas. The remaining mice were held for 30days in order to determine if the production of monoclonal antibodieswas successful.

Making Hybridomas

The protocol of “Standard Operating Procedure Production of MonoclonalAntibodies Fusion of Spleen Cells” was followed, relevant portions asfollows and is incorporated by reference in its entirety:

Procedure assumes that Balb/c mice have been immunized and boosted withantigen. 3-4 days prior to fusion one mouse has been given an IV boostwith antigen.

Materials

Cells: Fusion partner cell line—P3X63Ag8U.1 (X63)

Media and Additives:

-   1. Dulbecco's Modified Eagle's Medium with 4.5 g glucose (DMEM)-   2. L-glutamine (200 mM) or GlutMax-   3. Sodium pyuvate (100 mM)-   4. MEM Non Essential Amino Acids (100×) (NEAA)-   5. Penicillin-Streptomycin (10,000 units Penicillin/10,000 μg    Streptomycin)-   6. Fetal bovine serum—heat inactivated (FBS)-   7. Hypoxanthine/Thymidine (100×) (HT)-   8. Hypoxanthine/Aminopeterin/Thymidine (50×) (HAT)-   9. Polyethylene glycol 4000 (Sterile and tissue culture tested is    best from ATCC)-   10. Typan blue-   11. Hank's balance salts solution without calcium or magnesium

Plasticware (Sterile):

-   -   Pipets—1, 5, 10, 25 and 50 ml, 96-well flat bottom tissue        culture plates, Flasks—75 and 150 or 175 cm², Tubes—15 and 50 ml        screw cap tubes, Syringes—3 or 5 ml, Petri dishes 6 cm diameter,        Transfer pipets, Yellow pipet tips, Basins, Filters—1 L, 500 and        100 ml; bottle type with PES membrane

Equipment:

-   -   Waterbath set at 37 C, Tabletop centrifuge set at room        temperature ˜25 C, Autoclave, Hot plate, Microscope—bright field        for counting cells, Microscope—inverter, phase contrast for        observing cultured cells, Multi-channel pipettor—12 μlace 50-250        or 300 μl, Pipet-aid, Biological safety cabinet

Other:

-   -   Hemocytometer, 500 ml glass bottle to autoclave DI water, Screen        mesh for spleens—sterile, autoclaved, Forceps—small, sterile,        autoclave, Scissors—small, iris type, Test tube rack, 250 ml        glass beaker, Timer with seconds, 70% Isopropanol in a spray        bottle,

Preparation of Media

Media is prepares in a tissue culture filter apparatus. Some DMEM (˜70%of what is required) is added to the filter. The additives are addedusing an appropriate pipet. DMEM is added to approximately the finalvolume. The vacuum is applied. After the media goes through the filter,the filter is discarded and the lid is placed on the bottle. Media isgood for approximately 1 month at 4 C.

Media should be at 37 C when Used.

DMEM (Serum-Free):

-   -   (Used for washing spleen and myeloma cells and during the        fusion.) 250 ml/fusion    -   DMEM—235 ml, Glutamine—5 ml, Sodium pyruvate—2.5 ml, NEAA—2.5        ml, Penicillin/Streptomycin—2.5 ml, HT—2.5 ml

DMEM-HT:

-   -   (Used for growth of myeloma cells) 1 L    -   DMEM—850 ml, FBS—100 ml, Glutamine—10 ml, Sodium pyruvate—10 ml,        NEAA—10 ml, Penicillin/Streptomycin—10 ml, HT—10 ml

DMEM—20% FBS—HT:

-   -   (Used for growth of myeloma cells) 100 ml    -   DMEM—75 ml, FBS—20 ml, Glutamine—2 ml, Sodium pyruvate—1 ml,        NEAA—1 ml, Penicillin/Streptomycin—1 ml, HT—1 ml

DMEM-HAT:

-   -   (Used for growth of myeloma cells and the first day after        fusion) 1 L    -   DMEM—730 ml, FBS—200 ml, Glutamine—20 ml, Sodium pyruvate—10 ml,    -   NEAA—10 ml, Penicillin/Streptomycin—10 ml, HAT—20 ml        Steriled-filtered water 1 L—Needed during fusion.

Propagation of Myeloma Cell—X63

-   1. Place 30 ml of DMEM-HT in a 50 ml tube.-   2. Remove X63 cells from liquid nitrogen.-   3. Rapid thaw X63 cells by placing them in a 37 C waterbath.-   4. Spray with vial 70% isopropanol and place in BSC.-   5. After drying, open vial using a 2 ml pipet transfer the contents    to the centrifuge-   tube in step 1.-   6. Centrifuge at 1500 rpm (800×g) for 10 min.-   7. Remove supernatant and tap tube at the pellet to loosen.-   8. Add 15 ml of DMEM-HT. Mix.-   9. Transfer to 75 cm² flask. Place in incubator.-   10. Add media to cells when it begins to turn orange.    -   Need between 1-3×10⁸ cells for each fusion depending upon the        size of the spleen. Cells should be in log phase at the time of        fusion; just slightly orange, which is approximately 5×10⁵/ml.        This means that a minimum of 200 ml of cells are required for a        fusion.

Fusion Procedure Removal of Spleen

-   1. The mouse should be anesthetized by carbon dioxide gas and bled    by cardiac puncture. The mouse is euthanized by cervical    dissolocation.-   2. The mouse is sprayed with 70% ethanol or isopropanol and placed    in a BSC.-   3. Using sterile scissors and forceps, the skin is cut on the side    below the spleen (left side of mouse). The forceps are used to pull    back the skin and hair towards the head.-   4. Rinse the scissors and forceps with alcohol. Cut an incision the    body cavity to expose the spleen.-   5. Use new sterile small forceps and small scissors to remove the    spleen. Place in a tube containing Hank's balanced salts solution    (minus Ca and Mg). Place tube on ice.

Preparation of PEG

-   1. If the PEG is presterilzed, place it in a beaker of water that is    on a hot plate. The water should not cover the top of the PEG vial.    Heat the water to the PEG melts. If the PEG is not sterilized, weigh    out 1 g of PEG and place it in 13×100 mm screw cap glass tube.    Autoclave for 30 min on slow exhaust.-   2. Cool PEG by placing it in a beaker with water in a 37 C water    bath;-   3. Add 1 ml of warm DMEM-HT for each gram of PEG to the PEG. Place    back in water bath.    Place all media and sterile water in 37 C water bath.

Preparation of Lymphocytes

-   1. Place DMEM-HT in 37 C water bath prior to starting.-   2. In BSC, place screen mesh in bottom half of 6 cm Petri dish.-   3. Place spleen with Hank's in lid of Petri dish.-   4. Use sterile scissors and forceps to trim fat and connective    tissue from the spleen.-   5. Transfer the spleen to the Petri dish containing the screen. Add    DMEM-HT to the dish. Use 1 transfer pipet full.-   6. Use the top of the plunger of a 3 or 5 cc syringe to push and    grind the spleen into the screen. This breaks the spleen into single    cells and small pieces.-   7. Lift up the screen and wash it DMEM using a transfer pipet. Set    screen aside.-   8. Use a transfer pipet transfer the spleen cells to a 50 ml tube.    Rinse the Petri dish with DMEM and transfer to the tube.-   9. Allow debris to settle to bottom of the tube. Using a transfer    pipet, transfer the supernatant to a new 50 ml tube. Add DMEM to    40 ml. Discard debris tube.-   10. Centrifuge spleens cells 10 min at 1500 rpm (800×g).-   11. Pour off supernatant. Tap bottom of tuber to loosen pellet. Add    10 ml of DMEM-HT. Remove 100 μl for counting. Add 30 ml of DMEM-HT    to 50 ml tube containing spleen cells and centrifuge again as above.-   12. While centrifuging, take 10 μl of cells and mix with 90 μl of    trypan blue. Count cells in one large square of a hemocytometer.    Calculate total cells by multiplying the count by 10⁶.-   13. Pour off supernatant. Tap bottom of tuber to loosen pellet. Add    40 ml of DMEM-HT. Centrifuge as above with X63 cells from step 3    below.-   14. Remove spleen cells from centrifuge. Pour off supernatant. Tap    bottom of tuber to loosen pellet. Add 40 ml of DMEM-HT. Centrifuge    as above along with X63 cells in step 5 below.-   15. Remove spleen cells from centrifuge. Pour off supernatant. Tap    bottom of tuber to loosen pellet. Add 10 ml of DMEM-HT. These cells    get transferred to the tube containing the X63 cells in step 1 of    the fusion protocol.

Preparation of X63 Cells

-   1. Combine X63 cells into 1 flask. Remove 100 μl for counting.-   2. Add 100 μl of trypan blue to 100 μl of X63 cells. Place in    hemocytometer and count 1 large square. Determine cells/ml by    multiplying the count by 2×10⁴.-   3. Using the total spleen cell count from step 12 in section above    calculate the volume required to have the same number of cells as    spleen cells.-   4. Place the volume of cells in an appropriate number of 50 ml    tubes. Centrifuge in the same run as step 13 of the spleen cell    procedure.-   5. Pour off supernatant. Tap bottom of tuber to loosen pellet. Add    5-10 ml of DMEM-HT depending upon the number of tubes. Combine cells    into one tube with 40 ml of DMEM-HT. Centrifuge in the same run as    step 14of the spleen cell procedure.-   6. Pour off supernatant. Tap bottom of tuber to loosen pellet. These    cells get transferred to the tube containing the spleen cells in    step 1 of the fusion protocol.

Fusion Protocol

All media should be at 37 C.

-   1. Combine spleen cells and X63 cells in the same tube. Add DMEM-HT    to 40 ml.-   2. Centrifuge at 800 rpm (400×g) for 10 min. Aspirate supernatant.-   3. Place sterile 250 ml glass beaker in BSC. Add 100 ml of warm    sterile water to beaker.-   4. Place tube with pellet in the water.-   5. Place PEG/DMEM in BSC. Using a 1 ml pipet, pipet 1 ml of PEG.-   6. Add PEG to pellet slowly over 1 min with stirring. The tube is    held in the beaker of water.-   7. Stir for 1 more min.-   8. Using a 1 ml pipet, add 1 ml DMEM-HT over 1 min with stirring.-   9. Using a 1 ml pipet, add another 1 ml DMEM-HT over 1 min with    stirring.-   10. Over the next 2-3 min, add 7 ml of DMEM-HT with a 10 ml pipet    with stirring.-   11. Centrifuge at 800 rpm (400×g) for 10 min. Aspirate supernatant.-   12. Label 3 96 well flat bottom plates during the centrifugation.-   13. Add 10 ml of DMEM-20% FBS-HT by releasing in directly on the    pellet while stirring. There may be large clumps of cells. This is    fine.-   14. Add 20 ml of DMEM-20% FBS-HT and swirl the tube to resuspend    cells. Do not shake too hard, try to breakup clumps.-   15. Using a transfer pipet, distribute 0.1 ml per well. This is    approximately 2 drops. This should fill 3 plates. Do not use yellow    tips. The hole is too small and may disrupt the fused cells.

Feeding Schedule Day 0—Fusion day Day 1—Add 0.1 ml of DMEM-HAT Days 2,3, 5, 8, 11—

-   -   1. Remove 0.1 ml media per well with a 12 channel pipettor and        sterile yellow tips. Place spent media in a sterile basin. The        same tips can be used.    -   2. Place approximately 35 ml of DMEM-HAT in another sterile        basin. Add 0.1 ml media per well with a 12 channel pipettor and        sterile yellow tips. The same tips can be used.    -   3. Assay when the media in the wells starts to turn        orange/yellow. You may need to feed some individual wells sooner        than the schedule. The assay may also need to be done sooner        than the schedule. Remove 0.1 ml/well as in feeding and place in        assay plate. Add 0.1 ml of DMEM-HAT per well.    -   4. When viewed under the phase-contrast microscope, there should        be dieing spleen and non-fused X63 cells. There should also be        foci of fused cells that increase with time. They look similar        to the X63 cells that have not been exposed to HAT. They are        both attached to the bottom and in suspension.    -   5. If the cells are growing very fast, transfer them to 24 well        plates. Use autoclaved glass Pasteur pipets. The rubber bulbs        are soaked in 70% ethanol. 0.4 ml DMEM-HAT is added. This can be        increased to 1 ml and then 2 ml of DMEM-HAT.    -   6. The cells can be further transferred to 6 well plates, which        can take up to 10 ml media. The cells should be frozen and        cloned from these plates.    -   7. The cells can be slowly switched to DMEM-HT after transfer to        the 24 well plates if desired.

Testing of Antibody Production by ELISA

To prove the proper antibody production and select the wells for furthergrowing and cloning lipid and protein ELISAs were done (high cholesterolliposome w/o lipid-A, cholesterol, DMPC, DMPG and gp140).

Lipid ELISAs. The lipid ELISA was generally performed in accordance withthe methods described in:

-   Alving, B. M., Banerji, B., Fogler, W. E. and Alving, C. R. Lupus    anticoagulant activities of murine monoclonal antibodies to    liposomal phosphatidylinositol phosphate. Clinical and Experimental    Immunology 69 403-408 (1987), incorporated by reference:-   or, Swartz, Jr., G. M., Gentry, M. K., Amende, L. M.,    Blanchette-Mackie, E. J. and Alving, C. R. Antibodies to    cholesterol. Proceedings of the National Academy of Sciences, U.S.A.    85 1902-1906 (1988), Incorporated by reference with specific details    given below.

100 μl of cholesterol (5 nmol/well), DMPC (1 nmol/well), DMPG (10nmol/well) diluted in ethanol and 100 μl of high cholesterol-DMPC-DMPGliposome diluted in PBS were added into each well of Immulon 2HB Ubottom ELISA plate (Thermolab Systems) and allowed to dry in a hoodovernight. The cholesterol and liposome plates were blocked with 250 μlof blocking buffer (0.3% gelatin in PBS) and the DMPC, DMPG plates wereblocked with 250 μl of 3% BSA for 2 hours. Culture supernatant (50 μl)of each of the hybridomas was added to the plate at room temperature for2 hours. Plates were washed 5 times with washing buffer (20 mM Tris-HClpH 7.4, 154 mM NaCl) using an automated plate washer (Seltron MAPC) andexposed to the secondary antibodies as goat anti-mouse IgM antibodyconjugated to HRP (Zymed Labs) and sheep affinity-purified andHRP-linked anti-mouse IgG antibody (Binding Site Inc.) for 1 hour.Plates were washed, exposed to ABTS peroxidase substrate system (KPL) atroom temperature for 1 hour and then read at 405 nm with Spectra max 250(Molecular Devices).

Protein ELISA

0.1 ug of gp41, gp120 and gp140 diluted in 100 ul of PBS was added toeach well of Immulon 4HBX plates (Thermolab Systems) and allowed to dryin a hood overnight. The plates were blocked with 250 μl of blockingbuffer (0.5% casein and 0.5% BSA) for 2 hours. Culture supernatant ofthe given hybridomas was added to the plate at 4° C. for overnightincubation. Plates were washed 5 times with washing buffer (0.1%Tween-PBS) using an automated plate washer (Seltron MAPC) and exposed tothe secondary antibodies for 1 hour. Plates were washed, exposed to thesubstrate at room temperature for 1 hour and then read.

Cloning

Cloning was done twice by limiting dilution, and the clones were thentested by ELISA.

The MPR region 18 of gp41 as shown in FIGS. 2 and 3 contains the bindingepitopes for two human IgG monoclonal antibodies that are know to bebroadly neutralizing antibodies. They are known as 2F5 and 4E10. 2F5binds to ELDKWA (SEQ ID NO: 2) (the MPR starts at D) and 4E10 binds toNWFDIT (SEQ ID NO: 3). The 2F5 epitope, ELDKWA (SEQ ID NO: 2), is thesame sequence identified as the binding site for GalCer. The cholesterolbinding site LWYIK (SEQ ID NO: 4) is at the end of the MPR. The overallseries of interactions of HIV-1 involving budding, binding and fusionwith host and target cells exposes lipid-associated proteins, and evenlipids themselves, as targets for virus neutralization.

The proposed interactions of HIV-1 for fusion with the plasma membranelipid bilayer lipids are illustrated in FIG. 1. Plasma membraneglycosphingolipid microdomains as preferential sites of formation of theHIV-1 fusion complex. In the plasma membrane of CD4+cells, CD4 1 ispresent in glycosphingolipid enriched microdomains but is not associatedwith HIV-1 corereceptors. Once bound to CD4, the viral particle 2 isconveyed to an appropriate coreceptor 3 by the glycosphingolipid raft 4,which moves freely in the external leaflet of the plasma membrane 5.cholesterol 6; glycosphingolipid 4; phosphatidylcholine 7.

As shown in FIG. 2, after budding from host cells, the HIV-1 virus 2exhibits a strong tendency to infect T lymphocytes as target cells,using CD4 as a receptor 1 (Piguet & Sattentau, 2004). A is a crosssection of HIV-1 envelop protein. The binding and fusion of HIV with thetarget cell involves a choreographed ballet between the proteins of thefree virus 2 and the entry site of the target cell (B and C). HIV entryinto a cell is a multistep process initially involving the interactionsof viral envelope protein gp120 and gp41 with several binding sites onthe cell surface. The envelope proteins exist as a trimer consisting of3 gp120 molecules and 3 gp41 molecules. The binding of gp120 to CD4 isfollowed by conformational changes in the gp120 protein that exposebinding sites to chemokine receptors 3, CXCR4 or CCR5, that serve asco-receptor binding sites for interactions of the virus with the targetcell (Berger et al., 1999; Doms, 2000; Huang et al 2005). The binding ofgp120 to the chemokine co-receptor in turn induces conformationalchanges that allow the binding of the gp41 anchor protein to the cell,and this is followed by fusion of the viral lipid bilayer with theplasma membrane bilayer, and entry of the virion RNA into the targetcell (Colman & Lawrence, 2003) (C). The binding and entry processesentail numerous types of interactions between proteins and lipids of thevirus and specific lipids of the target cell (Fantini et al. 2002).

In FIG. 2, the reference numbers represent as follows: CD4 1, viralparticle 2, co-receptor 3, raft 4, plasma membrane 5, p17 matrix 10,lipid bilayer 11, membrane proximal region 18, fusion peptide 19. Alsostep 3 is fusion and entry.

Humans may be immunized with the appropriate liposomes to producemonoclonal antibodies that have broadly neutralizing activities and avaccine preparation can be made that would be composed of the aboveliposomal lipid and protein or peptide combination for testing forprotective efficacy against multiple types of HIV clades.

Safety of Antibodies to Lipids Generated by Liposomes Containing Lipid a

Preclinical studies demonstrated that life-long injection of liposomescontaining lipid A in mice induced antibodies against numerous lipids,but did not adversely affect the life span of the mice (Richardson etal., 1988-89). Moreover, life-long injection of saline alone into themice was also associated with age-related appearance of antibodies tolipids, but the antibodies did not appear to have caused any substantivedeleterious effects. Liposomes containing lipid A have proven to beextremely safe in numerous phase I and phase II experimental humanvaccine trials involving more than 200 volunteers. A high level ofsafety was observed even with very high concentrations of lipid A (Frieset al., 1992; Heppner et al., 1996; McElrath, 1995; Harris et al.,1999). In the 15 years that such clinical trials have been conductedthere have been no reported instances of association of the antibodieswith APS or any other autoimmune disease.

Under normal circumstances liposomes themselves are generally notconsidered immunogenic in experimental animals. However, by utilizinglipid A as an adjuvant in the lipid bilayer liposomes can be renderedimmunogenic, and antibodies to liposomal phospholipids have beenexperimentally induced in animals (rabbits and mice) (Schuster et al.,1978; reviewed by Alving, 1986). By using lipid A, which is the lipidmoiety of Gram-negative lipopolysaccharide (endotoxin), as an adjuvant,polyclonal and monoclonal antibodies to liposomal phospholipids (alsoreferred to as anti-phospholipid antibodies, or aPLs) (Banerji et al.,1982; Wassef et al., 1984); liposomal cholesterol (Swartz et al., 1988),and even liposomal squalene (an alkene precursor of cholesterol) (Matyaset al., 2000) have been developed.

Liposomes containing lipid A form the basis of the numerous proposedexperimental liposomal vaccines against diseases as diverse as malaria(Plasmodium falciparum) (Fries et al., 1992), HIV (McElrath, 1995; Raoet al., 2004; Richards et al., 2004), Ebola hemorrhagic fever (Rao etal., 2002),), ricin intoxication (Matyas and Alving, 1996), prostatecancer (Harris et al., 1999; Alving, 2002), and breast cancer (Samuel etal., 1998; Batenjany et al., 2001). In addition to production ofantibodies to liposome-encapsulated protein antigen that is present inthe vaccine formulation, it is possible that antibodies to the liposomallipids could also be induced by such vaccines. Considerable evidenceindicates that numerous types of circulating antibodies to lipids occurnaturally in virtually all normal humans and these antibodies generallydo not pose any recognized clinical risk.

Further, humans produce circulating antibodies against such fundamentalelements as lipids in the lipid bilayers or tissues of all mammaliancells but do not attack and damage those cells. This is because normalcells are protected from the binding of antibodies to lipids by sterichindrance from adjacent larger molecules.

EXAMPLES

The inventor has shown that certain types of liposomes have been shownto be synthetic models of stabilized lipid raft-like structures andimmunological studies with liposomes provide insights about interactionsof HIV-1 with lipid bilayers. The inventor found that HIV-1 lipidstructures, or liposomes, are useful either as antigens, or as auxiliarylipids in combination with proteins and peptides and have exploited themfor immunological approaches to HIV-1 and produced the following resultsas described in Examples 1 and 2. The following examples are presentedto illustrate the invention but it is not to be considered as limitedthereto.

Example 1

Antibodies have been produced that have subsite specificities both forlipid and amino acid epitopes and that will bind simultaneously to thelipids and the lipid-associated protein. Mice were immunized withliposomes containing lipid A and containing encapsulated protein fromthe envelope of HIV-1. A unique murine monoclonal antibody has now beengenerated that simultaneously recognizes both the envelope protein byitself, and also recognizes the protein-free liposomes by themselves. Inaddition, each of the antibodies reacts with at least one individualconstituent (cholesterol or high cholesterol liposomes, respectively)that was present in the immunizing liposomes. This demonstrates that theproduction of unique antibodies having such dual specificities canindeed be produced. Studies to determine whether this antibody hasneutralization activity against clinical isolates of HIV-1 are currentlybeing undertaken.

This work has resulted in immunization with synthetic liposomal lipidrafts containing gp140, and the identification and creation of threeclones that recognize both gp160 and either dimyristoylphosphatidylglycerol or cholesterol, or both as shown in FIGS. 5 a, 5 b,5 c, 5 d and 5 e.

FIGS. 5 a-5 e demonstrate the binding of five different clones thatrecognize either lipid alone (cholesterol, or high cholesterolliposomes) or protein (gp41 and gp140, but not gp120), or dualspecificity for both lipid and protein, as indicated.

The immunization consisted of the high cholesterol liposomes thatcontained encapsulated gp140. The gp140 protein consists of a proteinthat contains both gp120 and part of gp41; thus, the dual specificantibodies were specifically directed only to the gp41 portion of thegp140, and not to the gp120 portion.

Clone number Specificity 1 Cholesterol, and high cholesterol liposomes(i.e., lipid only) 2 gp41 and gp140 (i.e., protein only) 3 Highcholesterol liposomes, gp41, and gp140 (i.e., lipid and protein) 4. Highcholesterol liposomes, gp41, and gp140 (i.e., lipid and protein) 5.Cholesterol, high cholesterol liposomes, gp41, and gp140 (i.e., lipidand protein) *High Cholesterol liposome is a liposome with a cholesterolcontent of over 50%. The data as shown has had the low nonspecificbackground activities in the ELISA subtracted.ELISA subtracted.

These clones have, therefore, tentatively been found to bind to the mprregion of the gp 140, i.e. gp 41, they therefore, might have propertiesthat are similar to 2F5 or 4E10. Numerous other clones have also beenidentified which have the dual or multi-specific binding specificitiesdefined above. These further clones were obtained after immunizing withsynthetic lipid rafts containing phosphatidylinositol phosphate andmpr24, or V3 loop (P18) peptide together with galactosylceramide. Theresults from these tests are positive.

Example 2 Murine Monoclonal Antibody to Phosphatidylinositol Phosphate(PIP)

HIV-1 neutralizing capabilities of mabs to PIP have been tested.Antibodies to PIP have the ability to cross-react with cardiolipin whichis useful for testing the concept that antibodies to cardiolipin canhave broad neutralizing properties for HIV. Extensive experiments havenow demonstrated that monoclonal antibodies to PIP do exhibitneutralizing activities against two clinical field isolate strains ofHIV-1.

The murine monoclonal antibody to phosphatidylinositol phosphate (PIP)has been shown by the inventor to bind to PIP, as determined by ELISA.This anti-PIP antibody has been shown to have similar binding propertiesto human monoclonal antibody (4E10). Each of the antibodies had antigensubsite binding specificities in aqueous medium for smallphosphate-containing molecules and for inositol. The anti-PIP monoclonalantibody inhibited infection by two HIV-1 clinical isolates inneutralization assays employing primary human peripheral bloodmononuclear cells. The data suggest that PIP or related lipids havingfree phosphates could serve as targets for neutralization of HIV-1.

Recently, an important observation was made that two broadlyneutralizing human MAbs, known as 4E10 and 2F5, which are known to reactwith gp41 of HIV-1 envelope protein, cross-reacted with cardiolipin (CL)and are in the category of antibodies that have lupus anticoagulant-typeanti-CL specificities. This observation is also consistent with aprevious finding that HIV-1 could bind to, and fuse with, CL liposomes,and that such binding inhibited infection of A3.01 cells by HIV-1. Thelatter result suggested that HIV-1 has a binding site for CL. Theresults from the two laboratories could be interpreted as indicatingthat CL might serve as a binding site for HIV-1 and that interferencewith the binding to CL could be exploited for vaccine development.However, balanced against this, it is known that CL is not present as alipid constituent of either HIV-1 or of the plasma membrane of anymammalian cell, and this therefore raises the question whether analternative lipid antigen might actually be the real neutralizing, andperhaps more important, target of 4E10 and 2F5.

The inventor found that specific polyclonal and monoclonal antibodies tophosphatidylinositol-4-phosphate (PIP) can be readily induced in mice byinjection of liposomes containing PIP as an antigen and lipid A as anadjuvant (Alving 1986). Four complement-fixing murine monoclonalantibodies to PIP, selected for their ability to react with liposomescontaining PIP but not with liposomes lacking PIP, have been extensivelystudied (Alving 1987, Alving 1980, Alving 1986, Folger 1987, Friedman1982, Stollar 1989, Wasseff 1993). The anti-PIP antibodies arecharacterized by the ability to react with varied types ofphosphorylated molecules, including certain closely related anionicphospholipids that have charged non-zwitterionic phosphate groups, suchas CL (Alving 1987), and also with denatured DNA (Stollar 1989).Presumably because of cross-reactivity with CL, anti-PIP antibodies gavepositive results in clinical assays for lupus anticoagulant activity(Alving 1987). Anti-PIP antibodies can be inhibited by small solublephosphorylated molecules, such as inositol hexaphosphate (but notinositol), phosphocholine (but not choline), and nucleotides (but notnucleosides) (Alving 1987, Wassef 1993, Stollar 1989). Because of thephosphate-binding subsite that allows such haptenic inhibition to occur,the antibodies can actually serve as high affinity carriers and donorsfor biologically important molecules, as shown by ability of ATP boundto anti-PIP antibodies to serve as a high energy phosphate donor for anenzymatic (hexokinase) reaction.

In addition to providing information about the molecular architecture ofantigen binding subsites, Mabs to PIP are useful probes for exploringpotentially important biological binding and receptor activities.Anti-PIP antibodies bind directly to membrane phospholipid on adherentbut not on nonadherent macrophages. There is also evidence that PIP canbe expressed on the cell surface and act as a receptor for diphtheriatoxin. Antibodies to PIP inhibited diphtheria toxin-induced CHO cellcytotoxicity. In view of this, the inventor investigated the potentialrole that antibodies to PIP might play in the identification of targetphospholipid antigens for induction of effective neutralizing antibodiesto HIV. It was demonstrated that not only does the 4E10 antibodyresemble anti-PIP antibodies in that it binds to PIP and can beinhibited by small phosphorylated molecules, but specific monoclonalanti-PIP antibodies also resemble 4E10 in that they neutralize strainsof HIV-1, including two field isolates of HIV-1.

Murine monoclonal IgM antibodies to PIP were obtained after immunizingmice with liposomes containing PIP as an antigen and lipid A as anadjuvant, as previously described. IgM antibody was purified fromascites fluid containing anti-PIP antibody no. 4 by using the protocolsupplied with the ImmunoPure IgM Purification kit and Slide-A-Lyserdialysis cassettes (Pierce Chemical Co., Rockville, Ill.). Activities ofthe anti-PIP and 4E10 antibodies were assayed by ELISA, with slightmodification. Monoclonal antibody 4E10 was obtained through the NIH AIDSResearch and Reference Reagent Program. The methods for isolation,propagation, and titration of HIV-1 isolates, and the neutralizationassay, were used as previously described.

FIGS. 6 a, 6 b and 7 a, 7 b illustrate the binding of anti-PIP and 4E10antibodies to CL and PIP, as determined by ELISA, and the effects onbinding in the presence of soluble molecules containing free phosphategroups or inositol. The phosphate binding subsite of anti-PIP isrevealed by the ELISA data in that casein, a highly phosphorylatedprotein was inhibitory to binding of anti-PIP both to PIP and tocardiolipin, as was phosphocholine, but no inhibition was found withcholine (FIGS. 6 a,6B). The recently reported binding of the 4E10antibody to CL is confirmed by our data (FIG. 7 a), and a newspecificity of binding of 4E10 to PIP was also observed (FIG. 7 b). The4E10 antibody, as with anti-PIP, has a similar phosphate-binding subsitein that the binding to CL and PIP was inhibited by phosphocholine butnot by choline (FIG. 7 a, 7 b).

The binding of the anti-PIP and 4E10 antibodies to PIP was not inhibitedby soluble haptenic inositol (FIG. 7 a, 7 b), and inositol actuallyenhanced the binding of the antibodies to the antigen (FIG. 7 a, 7 b).No enhancement was observed in the binding of the antibodies to CL inthe presence of inositol (FIG. 6 a, 6 b). Separate experiments suggestedthat the enhancement of binding of the antibodies to PIP, but not to CL,probably reflects a low affinity hydroxyl-hydroxyl association ofinositol with the polyhydroxyl headgroups on PIP, combined with lowaffinity inositol subsites in the anti-PIP and 4E10 antibodies.

The murine anti-PIP monoclonal antibody was examined for possible HIV-1neutralizing activity in a model utilizing inhibition of infection ofprimary cultures of peripheral blood mononuclear cells. As shown inFIGS. 8 a (A) and (B), the anti-PIP antibody exhibited neutralizingactivity that blocked infection of PBMCs by both HIV strain 91US_(—)1(FIGS. 8 a (A)) and 00KE_KER2018 (FIG. 8 a (B)), both of which areprimary clinical isolate strains of HIV-1. Interestingly, when theantibodies were tested against pseudoviruses from multiple clades in theTZM-bl cell line model system, the 4E10 antibody exhibitedneutralization, but the anti-PIP antibody did not neutralize (data notshown). Blinded independent confirmation of the neutralizing activity ofthe anti-PIP antibody for blocking 91US_(—)1 infection of primary PBMCs(both with PBMCs from the donor shown in FIG. 8 a (A) and with PBMCsindependently obtained from a separate donor) was kindly provided by Dr.John Mascola at the Vaccine Research Center, NIH (data not shown).

Our data with murine Mab anti-PIP and human Mab 4E10, each of which bindto PIP and CL and neutralize HIV-1, suggest that cell-surface or viralPIP, or related inositol phosphatides, could play a role in theinteraction of HIV-1 with target cells. The inositol phosphatides, whichcomprise a family of eight chemical species with different combinationsof phosphate groups arranged around the polyhydroxyl inositol ring, arehighly versatile signaling molecules, with key roles inreceptor-mediated signal transduction, signal-induced actin assembly andremodeling, and membrane trafficking. PIP, an intermediate in thesynthesis of phosphatidylinositol-4,5-bis-phosphate (PIP2) fromphosphatidylinositol, is synthesized by a PI-4-kinase that is located inthe lipid rafts and caveolae-like vesicles of the plasma membrane ofeukaryotic cells. A huge and sometimes confusing array of proteins bindto inositol phosphatides, perhaps the most well-known of which areglycosylphosphatidylinositol (GPI)-anchored proteins. CertainGPI-anchored proteins, such as Thy-1 and CD59, are incorporated into theHIV-1 virion during budding of the virus from lipid rafts. GPI-anchoredproteins were originally discovered on the surface of Trypanosomabrucei, and antibodies to PI and PIP were induced in rabbits infectedwith Trypanosoma rhodesiense). Thus, PIP in an infectious organism canbe immunogenic, and it appears possible that PIP in the lipid bilayer ofthe HIV-1 virion or in the host cell, or other membrane lipids with freephosphate, could represent a target for neutralizing antibodies toHIV-1. Regardless of the exact mechanism of neutralization by anti-PIPand 4E10 antibodies, it does appear evident that the phosphate bindingsubsite, and possibly the inositol-binding subsite, of each antibodymight play a role. The data suggest that the neutralizing effects ofanti-PIP, and perhaps 4E10, may be more strongly associated with theheadgroups of the phospholipids than with hydrophobic interactions withthe hydrophobic regions of either HIV-1 or plasma membrane lipidbilayers.

ELISA Technique for PIP and Cardiolipin.

The ELISA procedure was developed by modification of previous techniquesfor analysis of antibody binding to lipid antigens (Smolarsky 1980;Loizou et al. 1985). PI, PS, or CL was coated onto the surface of wellsin polystyrene microtitre plates (Immunolon I, ‘U” bottom, Dynatech LabsInc., Alexandria, Va.) by addition of an ethonolic solution andevaporation of the solvent by air under a fume hood. Plates were furtherdried under high vacuum and stored at −20° C. when not used the sameday. Plates were blocked by addition of phosphate-buffered saline (PBS),pH 7.2, containing 0.3% gelatin (Difco Laboratories, Detroit, Mich.).This was accomplished by washing the wells three times for 5 min eachwith PBS containing 0.3% gelatin. Fifty microlitres of goat anti-mouseIgM (μ chain specific) alkaline phosphatase conjugate (Kirkegaard andPerry Laboratories, Inc. Gaithersburg, Md.) at 1 μg/ml in PBS containing1% BSA were added to the wells and incubated for 30 min. at 22° C.Plates were agains washed three times for 5 min each with PBS containing0.3% geletan. Fifty microlitres of the substrate p-nitrophenylphosphateat an initial concentration of 2 mg/ml in diethanolamine buffer9Kirkegaard and Perry Laboratories, Inc.) were added to the wells andincubated for 30 Min at 22° C. Plates were scanned for optical activityat 405 nm with Titertek Multiscan (Flow Laboratories, McLean, Va.).Values reported were adjusted by substracting values in wells thatlacked monoclonal antibody. Alving (1987)

Example 3

The inventor's further goal was to achieve a vaccine that would covernumerous clades, so they considered sequences that are highly conserved.Two such conserved antigenic regions are portions of the mpr region ofgp41 and the lipid bilayer itself, including lipids such asphosphatidylinositol phosphate, phosphatidylserine,phosphatidylglycerol, and cholesterol. Additionally, certain conservedregions in gp120, particularly conserved elements of the V3 loop, areknown to bind to glycolipids, including galactosyl ceramide andganglioside GM3.

The inventor has shown that a monoclonal antibody may be produced thathas successfully neutralized two field isolates of HIV-1 and a furthermonoclonal antibody that has been generated that simultaneouslyrecognizes both a protein (gp160 from HIV) and one or more protein-freeliposomal lipids. It would be understood by one of ordinary skill in theart that this method of producing a dual specific monoclonal antibodythat recognizes both an amino acid sequence and a lipid epitope can beapplied to other substances other than HIV-1. Other substances orentities to be neutralized are other viruses, bacteria, hormones, fungi,cancer cells, protozoa or any other entity that triggers an antibodyimmune response.

DEFINITIONS

4E10, 2F5, and Z13: These are designations of monoclonal antibodies,derived from individual humans infected with HIV-1, or that have beenidentified from phage display libraries, that have the ability tobroadly neutralize clinical isolates of HIV-1. The antibodies arefurther taught and described by Buchacher et al. [Buchacher A, Predl R,Strutzenberger K, Steinfellner W, Trkola A, Purtscher M, Gruber G, TauerC, Steindl F, Jungbauer A, Katinger H. Generation of human monoclonalantibodies against HIV-1 proteins; electrofusion and Epstein-Barr virustransformation for peripheral blood lymphocyte immortalization. AIDSResearch and Human Retroviruses. 1994 April; 10(4):359-369] and by Zwicket al. [Zwick M B, Labrijn A F, Wang M, Spenlehauer C, Saphire E O,Binley J M, Moore J P, Stiegler G, Katinger H, Burton D R, Parren P W,Broadly neutralizing antibodies targeted to the membrane-proximalexternal region of human immunodeficiency virus type 1 glycoproteingp41. Journal of Virology 2001 November; 75(22):10892-10905].Adjuvant: An adjuvant is defined as anything that will amplify theimmune response or improve the immune response over what the immuneresponse would be without the adjuvant.Antigenic epitope (or, more simply, an epitope): Molecular recognitionsite for binding of antibodies. Commonly this is determined or producedby injecting an antigenic material into a mammal, or by introduction ofthe antigenic material to lymphocytes in vitro, for presentation of theantigenic material to lymphocytes to induce antibodies that are secretedby lymphocytes, and said antibodies then have the capacity to bind tosites on the material that had been presented to the lymphocytes.Broadly neutralizing: A commonly encountered problem in HIV-1 immunologyand vaccinology is the inability of antibodies induced against HIV-1organisms produced in the laboratory to prevent (i.e., neutralize)primary isolates of HIV-1 viruses from infecting target cells. Broadlyneutralizing antibodies are defined as antibodies that have the abilityto partially or completely overcome this problem by neutralizing morethan one type of primary isolate of HIV-1 virus.Enveloped virus: A virus that has an envelope (i.e., an outer lipidbilayer structure together with associated proteins on the outersurface) is an enveloped virus. Examples of such viruses include: HIV-1,influenza virus, dengue virus, Sindbis virus, and Ebola virus, amongmany others.Dual-specific or multi-specific: This is defined as the ability of anantibody to bind simultaneously or independently to epitopes on two ormore types of antigenic chemical species, for example to an amino acidsequence and to a lipid; or to a sugar and a lipid; or to an amino acidsequence and a sugar. The term “dual” refers only to binding to morethan one type of chemical epitope, but such antibody bindingspecificities may actually contain as many molecular binding sites fordifferent types of chemical epitopes (including three, or more,epitopes) as there is available space on the binding site of theantibody for such simultaneous binding of more than one type of epitope.Lipid: Lipids are defined as taught by Small, D. M., “The PhysicalChemistry of Lipids, From Alkanes to Phospholipids” Handbook of LipidResearch, Vol, 4, Plenum, NY, 1986, p. 1, as given below:

-   -   “1.1 Definition of lipids: Assuming a broad definition, one can        define a lipid as any molecule of intermediate molecular weight        (between 100 and 5000) that contains a substantial portion of        aliphatic or aromatic hydrocarbon. Included are the        hydrocarbons, steroids, soaps, detergents, and more complex        molecules, such as triacylglycerols, phospholipids,        gangliosides, and lipopolysaccharides. Immediately, one can        imagine that the physical behavior of such chemically divergent        molecules will be quite different. Indeed one of the most        interesting characteristics of lipids is their tremendously        varied behavior in aqueous systems, ranging from almost total        insolubility (e.g., paraffin oil and sterol esters) to nearly        complete solubility (e.g., soaps, detergents, bile salts, and        gangliosides). This particular aspect of lipids is important        biologically because all cells exist in an aqueous milieu.”        Lipid structure (this includes all organized lipid structures,        or domains, and all solid phase, mesomorphic, crystalline,        liquid crystalline, and liquid lipid structures): This is        defined as all of the multiple organized physical states of        lipids, as taught by Small, D. M., in “The physical states of        lipids: solids, mesomorphic states, and liquids” in “The        Physical Chemistry of Lipids, From Alkanes to Phospholipids”        Handbook of Lipid Research, Vol, 4, Plenum, NY, 1986, Chapter 3,        pp. 43-87. All of the above terms are interchangeable as defined        in the context of this invention. Thus, the term “solid phase        lipid structure” is interchangeable with “mesomorphic states”,        “liquid lipids”, “organized lipid structures” “domains”,        “crystalline lipid structures”, liquid crystal lipid        structures”, and “liquid lipid structures”.        Lipid bilayer membrane: This is a type of double layer membrane        in which the polar groups of the parallel array of lipids of        each monolayer of lipids are oriented toward the aqueous phase        and the nonpolar groups (such as fatty acyl groups) of each        monolayer are oriented toward each other in the center of the        bilayer. Liposomes often contain lipid bilayers, as do plasma        membranes of cells.        Liposomes: Liposomes, as they are ordinarily used, consist of        smectic mesophases, and may consist or either phospholipid or        nonphospholipid smectic mesophases.

Definition of “Smectic Mesophase” as taught by Small, D. M., in “ThePhysical Chemistry of Lipids, From Alkanes to Phospholipids” Handbook ofLipid Research, Vol, 4, Plenum, NY, 1986, pp. 49-50 is given below:

-   -   “When a given molecule is heated, instead of melting directly        into an isotropic liquid, it may instead pass through        intermediate states called mesophases or liquid crystals,        characterized by residual order in some directions but by lack        of order in others . . . . In general, the molecules of liquid        crystals are somewhat longer than they are wide and have a polar        or aromatic part somewhere along the length of the molecule. The        molecular shape and the polar-polar, or aromatic, interaction        permit the molecules to align in partially ordered arrays . . .        . These structures characteristically occur in molecules that        possess a polar group at one end. Liquid crystals with        long-range order in the direction of the long axis of the        molecule are called smectic, layered, or lamellar liquid        crystals . . . . In the smectic states the molecules may be in        single or double layers, normal or tilted to the plane of the        layer, and with frozed or melted aliphatic chains.”        Primary isolates of HIV-1: These are isolates of HIV-1 that are        found spontaneously in human populations. Commonly, such        isolates are obtained from clinical specimens taken from        individuals naturally infected with HIV-1. Primary isolates        differ from latoratory isolates in that the latter are strains        of HIV-1 that are adapted to growth in transformed T cell lines.

Other bonding specificities of the antibodies of the invention are alsocontemplated. In addition to making dual specific antibodies,multi-specific antibodies for binding two or more antigenic epitopes arewithin the scope of the invention. These other antigenic epitopesinclude combinations of two or more amino acid sequences, lipids,sugars, and carbohydrates.

The invention has been described herein with reference to certainpreferred embodiments. However, as obvious variations thereon willbecome apparent to those skilled in the art, the invention is not to beconsidered as limited thereto.

REFERENCES

-   Aloia, R. C., Jensen, F. C., Curtain, C. C., Mobley P. W.,    Gordon, L. M., (1988) Lipid composition and fluidity of the human    immunodeficiency virus. Proc Natl Acad Sci USA 85:900-904.-   Aloia, R. C, Tian, H., and Jensen, F. C. (1993) Lipid composition    and fluidity of the human immunodeficiency virus envelope and host    cell plasma membranes. Proc. Natl. Acad. Sci. U.S.A. 90:5181-5185.-   Alving, B. M., Banerji, B., Folgler, W. E., and Alving C. R. (1987)    Lupus anticoagulant activities of murine monoclonal antibodies to    liposomal phosphatidylinositol phosphate., Clin. Exp. Immunol.    69:403-408.-   Alving, C. R., Iglewski, B., Urban, K. A., Moss, J., Richards, R.    L., and Sadoff, J. C., (1980) Binding of diphtheria toxin to    phospholipids in liposomes. Proc. Natl. Acad. Sci., U.S.A. 77:    1986-1990.-   Alving, C. R. (1986) Antibodies to liposomes, phospholipids, and    phosphate esters. Chem Phys Lipids 40:303-314.-   Alving, C. R. (2002) Design and selection of vaccine adjuvants:    Animal models and human trials, Vaccine 20:S56-S64.-   Banerji, B., Lyon, J. A., Alving, C. R. (1982) Membrane lipid    composition modulates the binding specificity of a monoclonal    antibody against liposomes. Biochim Biophys Acta, 689:319-326.-   Batenjany, M. M., Boni, L. T., Guo, Y., Neville, M. E., Bansal, S.,    Robb, R. J., Popescu, M. C. (2001) The effect of cholesterol in a    liposomal Muc 1 vaccine. Biochem Biophys Acta 1514:280-290.-   Berger, E. A., Murphy P. M. Farber, J. M. (1999) Chemokine receptors    as HIV-1 coreceptors: roles in viral entry, tropism, and disease.    Annu Ref Immunol. 17:657-700.-   Callahan, M. K., Popernack, P. M., Tsutsui, S., Truong, L.,    Schlegel R. A., Henderson, A. J., (2003) Phosphatidylserine on HIV    envelope is a cofactor for infection of monocytic cells. J. Immunol.    170:4840-4845.-   Chernomordik, L., Chanturiya, A. N., Suss-Toby, E., Nora, E.,    Zimmerberg, J. (1994) An amphipathic peptide from the C-terminal    region of the human immunodeficiency virus envelope glycoprotein    causes pore formation in membranes. J Virol. 68:7115-7123.-   Colman, P. M., Lawrence, M. C. (2003) The structural biology of type    I viral membrane fusion. Nat Rev Mol Cell Biol 4:309-319.-   Domas, R. W. (2000) Beyond receptor expression: the influence of    receptor conformation, density, and affinity in HIV-1 infection.    Virology 276:229-237.-   Fantini, J., Garmy, N., Mahfoud, R., Yahi, N. (2002) Lipid rafts:    structure, function and role in HIV, Alzheimer's and prion diseases,    Expert Rev Mol Med 20 December,    http://expertreviews.org/02005392h.htm.-   Fogler, W. E., Swartz, G. M., Alving, C. R. (1987) Antibodies to    phospholipids and liposomes: binding of antibodies to cells.    Biochim. Biophys. Acta 903:265-272.-   Freed, E. O., Martin, M. A. (1995) Virion incorporation of envelope    glycoproteins with long but not short cytoplasmic tails is blocked    by specific single amino acid substitutions in the human    immunodeficiency virus type 1 matrix. J Virol 69:1984-1989.-   Friedman, R. L., Inglewski, B. H., Roerdink, F.,    Alving, C. R. (1982) Suppression of cytotoxicity of diphtheria toxin    by monoclonal antibodies against phosphatidylinositol phosphate.    Biophys J 37:23-24.-   Fries, L. F., Gordon, D. M., Richards, R. L., Egan, J. E.,    Hollingdale, M. R., Gross, M., Silverman, C., Alving, C. R. (1992)    Liposomal malaria vaccine in humans: A safe and potent adjuvant    strategy. Proc. Natl. Acad. Sci. USA 89:358-362.-   Harris, D. T., Matyas, G. R., Gomella, L. G., Talor, E., Winship, M.    D., Spitler, L. E., Mastrangelo, M. J. (1999) Immunologic approaches    to the treatment of prostate cancer. Semin. Oncology 26:439-447.-   Heppner, D. G., Gordon, D. M., Gross, M., Wellde, B., Leitner, W.,    Krzych, U., Schneider, I., Wirtz, R. A., Richards, R. L., Trofa, A.,    Hall, T., Sadoff, J. C., Boerger, P., Alving, C. R., Sylvester, D.    R., Porter, T. G., Ballou, W. R. (1996) Safety, immunogenicity and    efficacy of Plasmodium falciparum repeatless circumsporozoite    protein vaccine encapsulated in liposomes, J. Infect. Dis.    174-361-366.-   Hill, C. P., Worthylake, D., Bancroft, D. P., Christensen, A. M.,    Sundquist, W. I. (1996) Crystal structures of the trimeric human    immunodeficiency virus type 1 matrix protein: Implications for    membrane association and assembly. Proc Natl Acad Sci USA    93:3099-3104.-   Huang, C. C., Tang, M., Zhang, M-Y., Majeed, S., Montabana, E.,    Stanfield, R. L., Dimitrov, D. S., Korber, B., Sodroski, J.,    Wilson I. A., Wyatt, R., Kwong, P. D. (2005) Structure of a    V3-containing HIV-1 gp120 core., Science 310:1025-1028.-   Matyas, G. R., Wasser, N. M, Rao, M., Alving, C. R. (2000) Induction    and detection of antibodies to squalenel, J Immunol Methods    245:1-14.-   McElrath, M. J. (1995) Selection of potent immunological adjuvants    for vaccine construction. Semin Cancer Biol. 6:375-385.-   Piguet, V., Sattentau, Q. (2004) Dangerous liaisons at the    virological synapse. J Clin. Invest 114:605-610.-   Rao, M., Bray, M., Alving, C. R., Jahrling, P., Matyas, G. R. (2002)    Induction of immune responses in mice and monkeys to Ebola virus    after immunization with liposome-encapsulated irradiated Ebola    virus: protection I mice requires CD4+T cells. J. Virol.,    76:9176-9185-   Rao, M., Matyas, G. R., VanCott, T. C., Birx, D. L.,    Alving, C. R. (2004) Immunostimulatory CpG motifs induce cytotoxic T    lymphocytes responses to human immunodeficiency virus type I    oligomeric gp140 envelope protein. Immunol. Cell Biol. 82-523-530.-   Richards, R. L., Rao, M., VanCott, T. C., Matyas, G. R., Birx, D.    L., Alving, C. R. (2004) Liposome-stabilized oil-in-water emulsions    as adjuvants: increased emulsion stability promotes induction of    cytotoxic T lymphocytes against an HIV envelope antigen. Immunol.    Cell Biol. 82:531-538.-   Richardson, E. C., Swartz, Jr., G. M., Moe, J. B., Alving, C. R.    (1988-89) Life-long administration of liposomes and lipid A in mice:    Effects on longevity, antibodies to liposomes, and terminal    histopathological patterns. J. Liposome Res. 1: 93-110.-   Samuel, J., Budzynski, W. A., Reddish, M. A., Ding, L.,    Zimmermann, G. L., Krantz, M. J., Koganty, R. R.,    Longenecker, B. M. (1998) Immunogenicity and antitumor activity of a    liposomal MUC1 peptide-based vaccine. Int. J. Cancer 75-295-302.-   Schuster, B., Neidig, M., Alving, B. M., Alving, C. R. (1979),    Production of antibodies against phosphocholine,    phosphatidylcholine, sphingomyslin, and lipid A by injection of    liposomes containing lipid A., J. Immunol. 122:900-905.-   Stollar, B. D., McInerney, T., Gavron, T., Wassef, N. M., Swartz    Jr., G. M., Alving, C. R. (1989) Cross-reactions of nucleic acids    with monoclonal antibodies to phosphatidylinositol phosphate and    cholesterol. Mol. Immunol. 26:73-79.-   Swartz Jr., G. M., Gentry, M. K., Amende, L. M.,    Blanchette-Mackie, E. J., Alving, C. R. (1988) Antibodies to    cholesterol, Proc Natl Acad Sci USA, 85:1902-1906.-   Trommeshauser, D., Krol, S., Bergelson, L. D., Galla, H. J. (2000)    The effect of lipid composition and physical state of phospholipids    monolayer on the binding and incorporation of a basic amphipathic    peptide from the C-terminal region of the HIV envelope protein gp41.    Chem Phys Lipids. 107:83-92.-   Wassef, N. M., Roerdink, R., Swartz, Jr., G. M., Lyon, J. A.,    Berson, B. J., Alving, C. R., (1984) Phosphate binding specificities    of monoclonal antibodies against phosphoinositides in liposomes.,    Mol Immunoll, 21:863-868.-   Wassef, N. M., Swartz, G. M., Alving, B. M., Alving, C. R. (1993)    ATP specifically bound as a hapten to a monoclonal anto-phospholipid    antibody retains phosphate donor activity. Biochem. Biophys. Res.    Commun. 190:582-588.-   Zwick M B, Labrijn A F, Wang M, Spenlehauer C, Saphire E O, Binley J    M, Moore J P, Stiegler G, Katinger H, Burton D R, Parren P W. (2001)    Broadly neutralizing antibodies targeted to the membrane-proximal    external region of human immunodeficiency virus type 1 glycoprotein    gp41. J Virol. 75:10892-10905.

What is claimed is:
 1. A method of making monoclonal antibodies thathave an antigen binding site that is dual- or multi-specific in bindingmore than one type of antigenic epitope comprising: a) obtaining anorganized lipid structure having a first lipid epitope and modifyingsaid organized lipid structure by incorporating (1) an adjuvant and (2)a protein or peptide epitope; b) immunizing a mammal with said organizedlipid structure; c) producing said antibodies, wherein said antibodieshave simultaneous recognition subsites to said lipid epitopes in saidorganized lipid structure and to said protein or peptide epitope; d)identifying said antibodies with simultaneous recognition subsites tosaid lipid epitopes in said organized lipid structure and to saidprotein or peptide epitope; e) isolating only said antibodies withsimultaneous recognition subsites to said lipid epitopes in saidorganized lipid structure and to said protein or peptide epitope; and f)cloning said antibodies to obtain monoclonal dual-multi specificantibodies having dual or multi-specific antigen binding sites bindingmore than one antigenic epitope selected from the lipid epitope and theprotein or peptide epitope.
 2. The method of claim 1, wherein said lipidand protein or peptide epitopes are from the same entity.
 3. The methodof claim 2 wherein said same entity is selected from the groupconsisting of viruses, bacteria, hormones, fungi, cancer cells andprotozoa.
 4. The method of claim 1, wherein said adjuvant is Lipid A. 5.The method of claim 1, wherein said organized lipid structure is a lipidor liposome.
 6. The method of claim 1, wherein said lipid epitopecomprise one or more of phosphatidylcholine, phosphatidylethanolamine,sphingomyelin, phosphatidylserine, phosphatidylinositol-4-phosphate,phosphatidylinositol, phosphatidyl glycerol, GalCer, SGalCer, CTH, GM1,GM3 and cholesterol.
 7. A method of making antibodies that have anantigen binding site that is dual- or multi-specific in binding morethan one type of antigenic epitope comprising: a) obtaining liposomeshaving a first antigenic epitope; b) modifying said liposomes byincluding a second antigenic epitope in said organized lipid structure;c) immunizing a mammal with said liposomes; d) producing saidantibodies, wherein said antibodies have simultaneous recognitionsubsites to said first antigenic epitopes in said liposome and to saidsecond antigenic epitopes; e) identifying said antibodies withsimultaneous recognition subsites to said first antigenic epitopes insaid liposome and to said second antigenic epitope; f) isolating onlysaid antibodies with simultaneous recognition subsites to said firstepitopes in said liposome and to said second antigenic epitope; g).cloning said antibodies to obtain monoclonal dual-multi specificantibodies having dual or multi-specific antigen binding sites bindingmore than one antigenic epitope selected from said first and secondantigenic epitopes.
 8. The method of claim 7, wherein said firstantigenic epitope and second antigenic epitope is selected from thegroup consisting of protein, peptide, polypeptide, amino acid sequence,lipid, sugar and carbohydrate.
 9. The method of claim 7, wherein saidlipid epitope comprise one or more of phosphatidylcholine,phosphatidylethanolamine, sphingomyelin, phosphatidylserine,phosphatidylinositol-4-phosphate, phosphatidylinositol, phosphatidylglycerol, GalCer, SGalCer, CTH, GM1, GM3 and cholesterol.
 10. Amonoclonal antibody comprising; an antibody having subsites thatsimultaneously recognize one or more epitopes selected from the groupconsisting of lipids, proteins, peptides, sugars, and carbohydrates. 11.A monoclonal antibody made by the process of claim
 1. 12. The monoclonalantibody of claim 10, wherein said protein or peptide epitopes are fromHIV-1 and comprise one or more of gp160, gp 140, gp120 and gp41.
 13. Themonoclonal antibody of claim 10, wherein said lipid epitopes compriseone or more of phosphatidylcholine, phosphatidylethanolamine,sphingomyelin, phosphatidylserine, phosphatidylinositol-4-phosphate,phosphatidylinositol, Phosphatidyl glycerol, GalCer, SGalCer, CTH, GM1,GM3 and cholesterol.
 14. The monoclonal antibody of claim 10, whereinsaid lipid epitopes comprise one or more of the lipid epitopes found ina lipid raft region of a plasma membrane of a host cell.
 15. A method ofmaking a monoclonal antibody to anionic phospholipids comprising:incorporating said anionic phospholipids and lipid A into a liposome;inserting said liposome into a mammal wherein said mammal producesmonoclonal antibodies to said phospholipids.
 16. The method of claim 15,wherein the anionic phospholipids is phosphatidylinositol phosphate(PIP).
 17. The method of claim 15, wherein said anionic phospholipids iscardiolipin.
 18. A method of making a monoclonal antibody tophosphatidylinositol phosphate (PIP) comprising: incorporating PIP andLipid A into a liposome; inserting said liposome into a mammal, whereinsaid mammal produces monoclonal antibodies to said PIP.
 19. A method ofbinding PIP antigen and or cardiolipin (CL) comprising: administeringanti-PIP antibody of claim 18 to a medium containing PIP antigen or CLantigen.
 20. A method of inhibition of infection of HIV-1 in primarycultures of peripheral blood mononuclear cells by HIV-1 comprisingadministering the anti PIP antibody of claim 18.